(1) General transformation techniques for micro-organisms
In recombinant DNA technology, transformation techniques for bacteria and yeasts are well developed, but transformation frequencies for moulds are relatively low.
For example, in genetic transformation of the bacterium Escherichia coli transformation frequencies of about 5.times.10.sup.8 transformants/.mu.g vector DNA have been obtained routinely, using a chemical transformation method. Approximately 3.5% of the viable cells became transformed (Hanahan; J. Mol. Biol. 166 (1983) 557-580). More recently, even higher frequencies, of 10.sup.9 to 10.sup.10 transformants/.mu.g vector DNA, have been reported after high voltage electroporation (Dower et al.; Nucleic Acids Research 16 (1988) 6127-6145). For other bacteria lower transformation frequencies have been described (e.g. Chassy and Flickinger; FEMS Microbiology Letters 44 (1987) 173-177; Miller et al.; Proc. Natl. Acad. Sci. USA 85 (1988) 856-860). For yeasts, transformation frequencies of up to 1.times.10.sup.7 transformants per .mu.g vector DNA have been obtained (Meilhoc et al.; Bio/Technology 8 (1990) 223-227 and Gietz et al.; Yeast 11 (1995) 355-360). For moulds, transformation frequencies vary from
only 0.1-0.5 transformants/.mu.g vector DNA for Agaricus bisporus (Van Rhee et al.; Mol Gen Genet 250 (1996) 252-258), via PA1 5 transformants/.mu.g vector DNA for Fusarium graminearum A3/5 (Royer et al.; Bio/Technology 13 (1995) 1479-1483), PA1 about 12 transformants/.mu.g vector DNA for Aspergillus awamori (Ward et al.; Experimental Mycology 13 (1989) 289-293), and PA1 20-300 transformants/ .mu.g vector DNA for Aspergillus nidulans (Yelton et al. Proc. Natl. Acad. Sci. USA 81 (1984) 1470-1474) to PA1 about 10.sup.4 transformants.mu.g vector DNA for Neurospora crassa (Volmer and Yanofsky; Proc. Natl. Acad. Sci. USA 83 (1986) 4869-4873). PA1 "Transformation in Fungi" by John R. S. Fincham published in Microbiological Reviews (March 1989) 148-170, which gives an outline of the possible transformation methods for fungi, i.e. both yeasts and moulds. PA1 "Genetic engineering of filamentous fungi" by Timberlake, W. E. and Marshall, M. A. Science 244 (1989) 1313-1317. PA1 "Transformation" by David B. Finkelstein (Chapter 6 in the book "Biotechnology of Filamentous Fungi, Technology and Products" (1992) 113-156, edited by Finkelstein and Ball).
For review articles on the transformation of moulds reference is made to the articles:
From this literature it is clear that several transformation techniques have been developed to transform an increasing number of filamentous fungi. Most transformation protocols make use of protoplasts. Protoplasts can be prepared from hyphal cultures or germinating conidia using Novozyme 234.sup.R, a multi-enzyme preparation derived from Trichoderma reesei. Transformation of protoplasts with DNA is mediated by electroporation or by a combination of CaCl.sub.2 and polyethylene glycol (PEG). Some alternative methods avoid the need for making protoplasts, which renders the procedure more rapid and simpler. Intact cells can be transformed using a combination of lithium acetate and PEG, particle bombardment (Lorito et al.; Curr. Genet. 24 (1993) 349-356 and Herzog et al.; Appl. Microbiol. Biotechnol. 45 (1996) 333-337) or also electroporation (Ozeki et al.; Biosci. Biotech. Biochem. 58 (1994) 2224-2227).
In view of the relatively low transformation frequencies of moulds in relation to the transformation frequencies of bacteria and yeasts, a need exists for higher transformation frequencies in moulds.
(2) Plant transformation using Agrobacterium
Another transformation technique developed for plants is based on the use of Agrobacterium tumefaciens, which is a gram-negative soil bacterium that causes crown gall tumors at wound sites of infected dicotyledonous plants. During tumor induction Agrobacterium attaches to plant cells and then transfers part of its tumor-inducing (Ti) plasmid, the transferred DNA or T-DNA, to the cell where it becomes integrated in the plant nuclear genome. The T-DNA is flanked by 24 basepair imperfect direct repeats. These direct repeats are also known as "border repeats" or "borders" or "T-DNA borders" or "border sequences" or combinations thereof. The T-DNA contains a set of genes. Expression of a subset of these genes, the onc genes, leads to the production of phytohormones which induce plant cell proliferation and the formation of a tumor. The process of transfer depends on the induction of a set of virulence genes encoded by the Ti plasmid. The transfer system is activated when VirA senses inducing compounds from wounded plants, such as acetosyringone (AS). Via the transcriptional activator VirG, the remaining vir loci are activated and a linear single-stranded DNA, the T-strand, is produced following nicking of the border repeats by a virD1/D2 encoded site-specific endonuclease. The VirD2 protein remains covalently attached to the 5' terminus. The T-strand is coated by the single-strand binding protein VirE and the resulting complex is transferred to the plant cell. Although the mechanism by which the T-DNA complex is transported from the bacterium into the plant cell is not well understood, it is thought that the T-DNA complex leaves the Agrobacterium cell through a transmembrane structure consisting of proteins encoded by the virB operon. For extensive reviews on Agrobacterium tumefaciens transformation see Hooykaas and Schilperoort (Plant Molecular Biology 19 (1992) 15-38) and Hooykaas and Beijersbergen (Annu. Rev. Phytopathol. 32 (1994) 157-179). The ability of Agrobacterium tumefaciens to transfer its T-DNA into the plant cell, where it is stably integrated into the nuclear genome, has lead to a widespread use of this organism for gene transfer into plants and plant cells. In order to allow the regeneration of plants after Agrobacterium tumefaciens transformation the onc genes in the T-region have been deleted, which resulted in a disarmed or non-oncogenic T-DNA. Two types of vector systems have been developed for plant transformation. First a binary system, in which new genes are cloned in between the T-DNA borders of a plasmid containing an artificial T-DNA This plasmid is subsequently introduced into an Agrobacterium strain harbouring a Ti plasmid with an intact vir region but lacking the T region (Hoekema et al.; Nature 303 (1983) 179-180 and Bevan; Nucl. Acids Res. 12 (1984) 8711-8721). Secondly a co-integrate system, in which new genes are introduced via homologous recombination into an artificial T-DNA already present on a Ti plasmid with an intact vir region (Zambryski et al.; EMBO-J. 2 (1983) 2143-2150).
A wide variety of plant species have been transformed using such systems. This includes many agriculturally important dicotyledonous species such as potato, tomato, soybean, sunflower, sugarbeet and cotton (for a review see Gasser and Fraley; Science 244, (1989) 1293-1299). Although Agrobacterium transformation of monocotyledonous plants seemed to be impossible for a long time, nowadays several species such as maize (Ishida et al.; Nature-Biotechnology 14 (1996) 745-750) and rice (Aldemita and Hodges; Planta 199 (1996) 612-617) have been transformed using Agrobacterium.
One of the reasons why the method has found wide use in plant transformation is its high transformation frequency. For instance in co-cultivation experiments with tobacco protoplasts about 25% percent of the microcalli, that were regenerated from protoplasts after co-cultivation with Agrobacterium (on average 20%), were transformed (Depicker et al.; Mol. Gen. Genet. 201 (1985) 477-484 and Van den Elzen et al.; Plant Molecular Biology 5 (1985) 149-154). This means that up to about 5% of the cells are transformed. Furthermore, the method is much easier compared with other plant transformation methods using naked DNA. It is applicable to intact plant tissues such as segments of leaves, stem, root and tubers as well as protoplasts. Additionally, the method has the advantage that only the T-DNA comprising the foreign DNA to be introduced is integrated into the plant genome. The vector DNA sequences required for replication and selection of the vector in the bacterium are not transported from the bacterium to the plant cell. Thus it is a relatively clean transformation method.
Another Agrobacterium species, Agrobacterium rhizogenes, possesses a similar natural gene transfer system.
(3) Transformation of micro-organisms using Agrobacterium
In addition to the many publications on transformation of plants using Agrobacterium tumefaciens, recently the results of some investigations on the use of Agrobacterium tumefaciens for transforming micro-organisms were published. Beijersbergen et al. (Science 256 (1992) 1324-1327) demonstrated that the virulence system of A. tumefaciens can mediate conjugative transfer between agrobacteria, which only relates to transformation of different strains of the same species. Bundock et al. (EMBO-J. 14 (1995) 3206-3214) reported on successful transformation of yeast by this soil bacterium. This result was subsequently confirmed by Piers et al. (Proc. Natl. Acad. Sci. USA, 93 (1996) 1613-1618). Both groups used DNA sequences from S. cerevisiae such as the yeast 2 .mu. origin (Bundock et al.; EMBO-J. 14 (1995) 3206-3214) or yeast telomeric sequences and the ARS1 origin of replication (Piers et al.; Proc. Natl. Acad. Sci. USA, 93 (1996) 1613-1618) in order to stabilize the T-DNA in yeast. Very recently, Risseeuw et al. (Mol. Cell. Biol. 16 (1996) 5924-5932) and Bundock & Hooykaas (Proc. Natl. Acad. Sci. USA, 93 (1996) 15272-15275) reported results on the mechanism of T-DNA integration in S. cerevisiae.
The data made available by these publications show that the transformation of micro-organisms by Agrobacterium tumefaciens is much less effective than that of plants. As mentioned above, in plants up to about 5% of the cells have been transformed, whereas for yeast much lower ratios of transformed cells/recipient cells are reported, namely 3.times.10.sup.-3 (Piers et al., Proc. Natl. Acad. Sci. USA, 93 (1996) 1613-1618) and 3.3.times.10.sup.-6 (Bundock et al. EMBO-J 14 (1995) 3206-3214).
Additionally, A. tumefaciens transformation of micro-organisms proved to be less efficient than traditional transformation techniques for micro-organisms. Usually the transformation frequency for naked DNA transfer is depicted as the number of transformants per .mu.g vector DNA, whereas the transformation frequency for A. tumefaciens transformation is often expressed as the number of transformed cells that can be obtained in relation to the number of recipient cells. In a prior publication on conventional transformation of yeast (Gietz et al.; Yeast 11, (1995) 355-360) both figures on transformants/ .mu.g vector DNA and figures on transformed cells per recipient cell are given, which gives a link between the two methods of calculating the transformation frequency.
Gietz et al. determined that with their LiAc/SS-DNA/PEG procedure a maximum of about 4% of the yeast cells in the reaction could be transformed, i.e. a transformation frequency of up to 4.times.10.sup.-2. From FIG. 1A and the corresponding description of this publication one can calculate that this 4% corresponds with 8.times.10.sup.5 transformants/.mu.g vector DNA. For A. tumefaciens transformation of yeast the maximal reported transformation frequencies are 3.times.10.sup.-3 (Piers et al.; Proc. Natl. Acad. Sci. USA, 93 (1996) 1613-1618) and 3.3.times.10.sup.-6 (Bundock et al.; EMBO-J. 14 (1995) 3206-3214), which is a factor of about 10 or 10,000, respectively, lower than the maximum transformation frequency (4%) of yeast with naked DNA reported by Gietz et al. Thus based on this evidence A. tumefaciens does not seem to be an additional promising tool for the transformation of micro-organisms, because the transformation frequencies obtained with A. tumefaciens are much lower than with the conventional transformation methods of yeast.